Negative regulation of defense responses in plants by a conserved MAPKK kinase

Abstract

The enhanced disease resistance 1 (edr1) mutation of Arabidopsis confers resistance to powdery mildew disease caused by the fungus Erysiphe cichoracearum. Resistance mediated by the edr1 mutation is correlated with induction of several defense responses, including host cell death. Double mutant analysis revealed that all edr1-associated phenotypes are suppressed by mutations that block salicylic acid (SA) perception (nim1) or reduce SA production (pad4 and eds1). The NahG transgene, which lowers endogenous SA levels, also suppressed edr1. In contrast, the ein2 mutation did not suppress edr1-mediated resistance and associated phenotypes, indicating that ethylene and jasmonic acid-induced responses are not required for edr1 resistance. The EDR1 gene was isolated by positional cloning and was found to encode a putative MAP kinase kinase kinase similar to CTR1, a negative regulator of ethylene responses in Arabidopsis. Taken together, these data suggest that EDR1 functions at the top of a MAP kinase cascade that negatively regulates SA-inducible defense responses. Putative orthologs of EDR1 are present in monocots such as rice and barley, indicating that EDR1 may regulate defense responses in a wide range of crop species.

Figure 1

Suppression of the edr1 resistance phenotype by mutations that block SA-mediated defense responses. Double mutants containing edr1 and the indicated mutations were constructed by standard genetic crosses. Plants were inoculated with E. cichoracearum and disease phenotypes scored 8 days after inoculation. Single representative leaves were removed from intact plants on the eighth day for photography. In the wild-type and double mutant plants, except for ein2/edr1, abundant asexual sporulation is observed as white patches. Also note the absence of yellow/brown necrotic patches observed in the edr1 mutant leaf.

Figure 2

Positional cloning of the EDR1 gene. (A) Genetic and physical map of the region flanking EDR1. The vertical dotted lines indicate the position of the closest markers that defined the edr1 genetic interval. Sequencing of three candidate genes in this interval revealed a C→G transversion in gene F22013.21, which produced an early stop codon. Comparison of the genomic sequence to the cDNA sequence obtained by reverse transcription–PCR revealed the indicated exon (boxes)-intron (lines) structure. (B) Complementation of the edr1 mutation by Agrobacterium-mediated transformation. Shown are sibling plants from the T2 generation that lack the wild-type EDR1 transgene (Left) or that contain the EDR1 transgene (Right). The apparent difference in size is because of necrosis of the larger leaves in the edr1 mutant (resistant) plant.

Figure 3

EDR1 encodes a MAPKKK with high similarity to CTR1 and four additional kinases present in Arabidopsis. (A) The predicted EDR1 protein contains 933 amino acids, the carboxyl-terminal third of which contains a kinase domain. (B) RNA gel blot analysis of the EDR1 mRNA. Wild-type and edr1 mutant plants were inoculated with E. cichoracearum (+) or mock inoculated (−) and poly(A) RNA isolated from leaves 3 days after inoculation. The blot was hybridized with an EDR1 cDNA probe corresponding to amino acid residues 180–461. Relative amounts of RNA loaded in each lane were estimated by using an EF-1α cDNA probe. PhosphorImager quantitation of this blot, normalized to the EF-1α signal, revealed that the edr1 message is induced approximately 5-fold by infection with E. cichoracearum. This analysis was repeated three times with similar results. (C) Alignment of the kinase domains of the six known members of the CTR1 kinase family in Arabidopsis. The entire protein for each of the indicated proteins was aligned by using the default parameters of clustalx, a Macintosh version of the clustalw program (47). Only the kinase domain is shown. The full-length alignment can be viewed in supplemental Fig. 6. The alignment file produced by clustalx was formatted by using the boxshade www server (http://www.ch.embnet.org/software/BOX_form.html). White type on a black background indicates a residue that is identical in at least half of the proteins shown, whereas a gray background indicates conservative substitutions as defined by the boxshade 3.21 default parameters. Asterisks indicate residues that are conserved in nearly all protein kinases (48). Arrows indicate regions conserved in the Arabidopsis, tomato, and rice EDR1 orthologs (see supplemental Fig. 6) but divergent in paralogs. Accession nos. for CTR1 and EDR1 are A45178 and AF305913, respectively. Accession no. AC034107 refers to the BAC DNA sequence, from which we derived the indicated protein sequence by using the EDR1 and CTR1 sequences as guides.

Figure 4

Ethylene sensitivity of edr1 mutant plants. (A) Ethylene-induced senescence. Wild-type and edr1 mutant plants were grown in pots for 6 weeks in a growth cabinet set for 9-h days then placed in a sealed chamber containing 100 ppm ethylene. Plants were removed for photography after 72 h. Note the increase in the number of yellow leaves in the ethylene-treated edr1 mutant. (B) Quantification of chlorophyll levels. Leaves 3 through 8 (leaf 1 being the oldest true leaf) were removed from 6-week-old plants after 72 h of exposure to 100 ppm ethylene and chlorophyll levels (microgram/gram fresh weight) measured as described (5). Bars represent the mean and standard deviation of values obtained from six plants. (C) Ethylene-induced expression of PR-1. RNA gel blots were performed with 10 μg of total RNA isolated from rosette leaves (both green and yellow) after 72 h of exposure to ethylene (+) or air (−). Blots were sequentially probed with PR-1 and EF-1α. The latter demonstrates approximately equal RNA loading in all lanes.

Figure 5

Conservation of EDR1 among diverse angiosperms. Full-length protein sequences were aligned by using the clustalw program (see supplemental Fig. 6) and a phylogenetic tree derived by using the neighbor-joining method (47). Numbers indicate bootstrap values supporting branch points based on 1,000 replicates. T09911, CAB87658, ACO34107, and AAF24836 are Arabidopsis CTR1/EDR1 homologs of unknown function. Putative EDR1 and CTR1 orthologs are preceded by a two-letter prefix to indicate species of origin: Hv, Hordeum vulgare (barley), Ri, O. sativa (rice), Le, Lycopersicon esculentum (tomato), At, A. thaliana.